Basic osteoblast growth factor II (bOGF-II)

ABSTRACT

A protein is provided derived from human fibroblast having an N-terminal amino acid sequence represented by SEQ ID NOS: 1 and 2, having a molecular weight of approximately 15kD under reducing and non-reducing conditions by (dosium dodecyl sulfate-polyacrylamide gel electrophoresis), and having an activity to stimulate growth of osteoblast. The protein is produced by gene engineering procedures, contains the amino acid sequence represented by SEQ ID NO: 9 and has osteoblast growth activity. Also provided is a method for preparing the protein by culturing human fibroblast and treating the conditioned medium for purification. The protein is utilized for the treatment of diseases characterized by decreased bone mass such as osteoporosis or is utilized as an antigen for immunological diagnosis of diseases.

RELATED APPLICATION

This application is a national stage application of International (PCT)Application JP95/01270 filed Jun. 26, 1995.

FIELD OF THE INVENTION

This invention relates to a novel protein, basic osteoblast growthfactor II (bOGF-II) which stimulates osteoblast growth, and methods forproducing the protein.

BACKGROUND ART

Human bones are always remodelling by the repeated process of resorptionand reconstitution. In the process, osteoblasts and osteoclasts areconsidered to be the calls mainly in charge of bone formation and boneresorption, respectively. A typical example of disease caused byabnormal bone metabolism proceeded by the bone cells is osteoporosis.The disease is known to be provoked by the condition in which boneresorption by osteoclasts exceeds bone formation by osteoblasts, but themechanism of osteoporosis has not yet been completely elucidated.Osteoporosis causes pain in the bone and makes the bone fragile, leadingto fracture. Since osteoporosis increases the number of bedridden oldpeople, it has become a social issue with the increasing number of oldpeople. Therefore, efficacious drugs for the treatment of the diseaseare expected to be developed. Bone mass reduction caused by the abnormalbone metabolism is thought to be treated by inhibiting bone resorption,improving bone formation, or improving the balanced metabolism.

Bone formation is expected to be promoted by stimulating growth,differentiation, or activation of osteoblasts. Recently, cytokines whichstimulates growth or differentiation of osteoblasts have been attractedpublic attention and have been intensively studied. Many cytokines arereported to stimulate the growth of osteoblasts, i.e. fibroblast growthfactor (FGF) (Rodan S. B. at al., Endocrinology vol.121, p1917, 1987),insulin-like growth f actor-I (IGF-I) (Hock J. M. et al., Endocrinologyvol. 122, p254, 1988), insulin-like growth factor-II (IGF-II) (McCarthyT. et al., Endocrinology vol.124, p301, 1989), and bone morphogeneticprotein (BMP) (Sampath T.K. etl al., J. Biol Chem. vol.267, p20532,1992, Knutsen R. et al., Biochem. Biophys. Res. Commun. vol.194, p1352,1993, and Akira Yamaguchi et al., Zikken Igaku vol.10, p2003, 1992).Many cytokines are also reported to stimulate the differentiation ofosteoblasts, i.e. transforming growth factor-s (TGF-P) (Centrella M. etal., J. Biol. Chem. vol.262, p2869, 1987), insulin-like growth factor(IGF), and bone morphogenetic protein (Takuwa :Y. et al., Biochem.Biophys. Res. Commun. vol.174, p96, 1991, and Knutsen R. et al.,Biochem. Biophys. Res. Commun. vol.194, p1352, 1993). These cytokinesare expected to be efficacious drugs for improving bone mass bystimulating bone formation; some of the cytokines such as bonemorphogenetic proteins are now investigated in clinical trials for theireffects to cure the patients with bone diseases.

Examples of drug products now clinically utilized for the treatment ofbone diseases and for shortening the treatment period are dihydroxyvitamine D₃, calcitonin and its derivatives, hormones such as estradiol,lprif lavon, and calcium preparations. However, these drug products donot provide satisfactory therapeutic effects, and novel drug substanceshave been expected to be developed. As mentioned, bone metabolism iscontrolled in the balance between bone resorption and bone formation.Therefore, cytokines which stimulate osteoblast growth and osteogenesisare expected to be developed as drug for the treatment of bone diseasessuch as osteoporosis.

Disclosure of the Invention

The inventors have intensively searched for osteoblast growth factors,and have found a novel osteoblast growth factor. The inventors have alsoestablished methods for accumulating the protein in a high concentrationand purifying it efficiently.

A cDNA clone encoding this protein was isolated by using the partialamino-acid sequences of the native bOGF-II protein. Moreover, bOGF-IIwas produced by the genetic engineering techniques with this cDNA. Theobject of the invention is to provide a novel osteoblast growthfactor.(protein) and methods for efficiently producing the protein.

The inventors screened animal cells conditioned media, and have foundthe osteoblast growth factor in human fibroblast IMR-90 (ATCC-CCL186)conditioned medium. The inventors examined the culture conditions ofIMR-90 cells, and have established the method for culturing the cells onalumina ceramic fragments to accumulate the osteoblast growth factor ina high concentration in the culture medium. The inventors found themethod to purify bOGF-Il efficiently by a combination of ion-exchangecolumn and/or heparin column.

Moreover, the inventors determined the amino acid sequences of thebOGF-II protein, designed the primers based on these amino acidsequences, and obtained a cDNA fragments of bOGF-II from a cDNA libraryof IMR-90 cells.

A cDNA clone encoding the full length protein of the current inventionwas isolated from a cDNA library of IMR-90 cells by hybridization usingthe cDNA fragment as a probe. Furthermore, the inventors established amethod for producing recombinant bOGF-II in the culture media of thecells which was transformed by expression vector containing the cDNA.

This invention relates to a protein characterized by the followingfeatures: (1) derived from human fibroblast cells, (2) molecular weightof ca. 15kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) under reducing and non-reducing conditions, (3) a highaffinity for cation exchanger and heparin, (4) decrease in osteoblastgrowth activity by heating at 70° C. for 10 minutes, and (5)inactivation by heating at 90° C. for 10 minutes. bOGF-II in the presentinvention is apparently different from known osteoblast growth factorsin N-terminal amino acid sequence. The N-terminal amino acid sequencesof bOGF-II are shown in Sequence Number 1 and 2.

The invention also relates to a method for producing bOGF-II,comprising: (1) culturing human fibroblasts, (2) treating the culturemedium through a heparin-column, (3) eluting an adsorbent fraction, (4)treating the eluate through an anion-exchange column to obtain anon-adsorbent fraction, (5) applying the fraction to a cation-exchangecolumn, and (6) purifying the objective protein through aheparin-column.

Purification procedure according to the invention includes any meanshaving the same effect as that obtained by the method for mixing aculture medium with heparin-Sepharose, etc. in a batchwise operation andfor treating through a column, as well as a simple method for flowing aculture medium through heparinSepharose column, etc.

bOGF-II can be efficiently isolated from a culture medium of humanfibroblasts at a high yield according to the invention. Isolation ofbOGF-II is based on general means for purifying proteins frombiomaterials, utilizing physical and chemical properties of theobjective protein bOGF-II. For example, concentration procedure includesgeneral biochemical technique such as ultrafiltration, lyophilization,and dialysis. Purification procedure includes combinations of severaltechnique for purifying proteins, such as ion-exchange chromatography,affinity chromatography, gel filtration chromatography, hydrophobicchromatography, reversed-phase chromatography, and preparative gelelectrophoresis. Human fibroblast is preferably IMR-90. The culturemedium of human fibroblast cell IMR-90 is obtained by absorbing humanfibroblast cell IMR-90 on ceramic, and culturing in DMEM mediumsupplemented with 5% fetal calf serum in a roller bottle in stationarystate for about a week. For purification, 0.1% CHAPS(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate) ispreferably added to a buffer as surfactant.

The protein of the invention is purified by applying the culture mediumto a heparin-column (heparin-Sepharose CL6B, manufactured by Pharmacia),eluting with 10 mM Tris-HCl buffer containing 2 M NaCl, pH 7.5 applyingthe diluted fraction to anion-exchange column (Hiload-Q/FF, manufacturedby Pharmacia), collecting an non-adsorbent fraction, and applying theobtained fraction to S cation-exchange column (Hiload-S/HP, manufacturedby Pharmacia) to fractionate three peaks, bOGF-I (0.15 M NaCl), bOGF-II(0.35 M NaCl), and -bOGF-III (0.55 M NaCl) in the order that theactivity is eluted in lower concentrations of NaCl. bOGF-II can beisolated by the following repeated heparin column chromatography(heparin-5PW, manufactured by Toso Co.), and can be identified by thepreviously described properties. It is likely that the protein bOGF-IIIis basic fibroblast growth factor from the results that it isinactivated by heating at 70° C. for 10 minutes, it is eluted by ca. 1.8M NaCl from heparin 5PW column, and it is inactivated by anti-basicfibroblast growth factor antibody.

Furthermore, a method for producing recombinant bOGF-II was established.The method includes the following three steps; First, amino-acidsequences of bOGF-II are used to design oligonucleotide primers. Second,a bOGF-II cDNA fragment is obtained by PCR amplification using theprimers. Finally, the cDNA clone encoding the full length bOGF-II isisolated from a cDNA library of IMR-90 cells by hybridization using thecDNA fragment as a probe. Moreover, bOGF-II is recovered and purifiedfrom culture medium or the cells by culturing host cells selected fromeukaryote such as mammalian cells (e.g. chinese hamster ovary cell) orprokaryote such as bacteria (e.g. E. coli.), which are transfected byvector, having expression promoter and the cDNA coded full-lengthbOGF-II.

The invention relates to proteins having an activity to stimulate growthof osteoblasts, containing the described amino acid sequence as a part,or having a homology with the described amino acid sequence more than801, and cDNA of the protein.

OGF activity can be evaluated by utilizing osteoblastic cell lines ornormal asteoblasts as target cells and by measuring an increasedincorporation ³ H-thymidine to the cells. The target cell is preferablymouse osteoblastic cell line MC3T3-E1 (J. Oral. Bio. Cell. Biol. 96,191, 1983). The cell is reported to be responsive to vitamin D₃ andparathyroid hormone and to grow up to be calcificated in vitro in amanner similar _(to) that in vivo. The OGF activity is preferablymeasured with a serum-free medium, and can be exactly evaluated at highsensitivity by measuring incorporation of ³ H-thymidine.

bOGF-II is useful as a pharmaceutical composition for treating orimproving decreased bone mass such as in osteoporosis and other diseaseswith abnormal bone metabolism, or as antigen for immunological diagnosisof the diseases.

bOGF-II is formulated to be pharmaceutical preparation, and can beorally or parenterally administered. The preparation comprises bOGF-IIas a an effective ingredient, and is safely administered to humanbeings. Examples of the pharmaceutical preparation include compositionsfor injection or intravenous drip, suppositories, nasal preparations,sublingual preparations, and tapes for percutaneous absorption. Thepreparation for injection is a mixture of bOGF-II in pharmacologicaleffective amount and pharmaceutically-acceptable carrier. The carrier isvehicle/activator which is generally added to compositions forinjection, e.g. amino acids, saccharides, cellulose derivatives, andother organic/inorganic compounds. When mixing bOGF-II with thevehicle/activator to prepare injections, pH adjustor, buffer,stabilizer, solubilizing agent, etc. may be added as needed.

BRIEF DESCRIPTION OF DRAWING

FIG. 1 shows elution pattern of bOGF fractions on a cation-exchangecolumn (HiLoad-S/HP™, 2.6×10 cm, manufactured by Pharmacia Co.). In theFIG. 1, peak 1 indicates bOGF-I, peak 2 indicates bOGF-II, and peak 3indicates bOGF-III.

FIG. 2 shows elution pattern of bOGF-II fraction on an affinity column(Heparin 5PW TM, 0.8×7.5 cm, manufactured by Toso Co.).

FIG. 3 shows pattern of bOGF-II on sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) under non-reducing condition.

In the FIG. 3, lane 1 indicates molecular weight marker, lane 2indicates fraction A, lane indictes fracton B, lane 4 indicates fractionC, lane 5 indicates fraction D, and lane 6 indicates fraction E.

See FIG. 2 for the fractions A-E.

FIG. 4 shows elution pattern of the peptides from reduced PE bOGF-IIwhich was digested with endoproteinase Asp-N (manufactured by BayringerCo.) on a reversed-phase column (OD-300, C18, 2.1×200 mm, manufacturedby Applied Biosystems Co.).

FIG. 5 shows activity of bOGF-II tested with MC3T3-E1.

FIG. 6 shows the structure of plasmid pQE30-OGF-II. In this drawing,6His represents histidine cluster; Phage T5 promoter represents phage T5promoter; bla represents an ampicillin resistant gene; Orirepresents-replication origin in E. coli and bOGF-II represents OGF-IIcDNA, respectively.

FIG. 7 shows activity of His6-bOGF-II tested with MC3T3-E1.

In the drawing, column 1 shows a control and column 2 shows the activitywhen a 10% solution of Hls6-bOGF-II is added.

THE BEST MODE TO CONDUCT THE INVENTION [Examples]

Detailed description of the invention is provided by way of examples asfollows. However, it should be noted that the examples are simplyillustrative, and the invention is not restricted to them.

Example 1 Production of natural type of bOGF-II

(1) Preparation of a conditioned medium of human fibroblast IMR-90

Human fetal lung fibroblast IMR-90 (ATCC-CCL186) was cultured on aluminaceramic fragments (80 g) (alumina: 99.5%, manufactured by T oshibaCeramic K.K.) in DMEM medium (manufactured by Gibco Co.) supplementedwith 5% FCS and 10 mM HEPES buffer (500 ml/roller bottle) in stationarystate at 37° C. in the presence of 5% CO₂ for 7 to 10 days using 60roller bottles (490 cm², 110×171 mm, manufactured by Coning Co.). Theconditioned medium was harvested, and a fresh medium was added to obtain30L of IMR-90 conditioned medium in one batch of culture. The obtainedconditioned medium was designated as sample 1.

(2) A method for testing osteoblast growth activity

Activity of osteoblast growth factor was evaluated by measuring DNAincorporation to mouse osteoblast MC3T3-E1 (granted by Dr. MasayoshiKumegawa, Professor of the Department of Dentistry at MeikaiUniversity). Precisely, sample solution (50 μl) which was diluted withα-MEM medium (manufactured by Gibco Co.) containing no nucleic acids andsupplemented with 0.2% BSA, was transferred to a 96-well microplate.Next, MC3T3-El cells in the α-MEM medium were inoculated to themicroplate at 2×10³ cells/50 μl and were cultured at 37° C. in 5% CO₂air for 15 to 20 hours. After the culture, 10 μl of ³ H-thymidine(TRK686, manufactured by Amasham Co.) diluted with phosphate bufferedsaline to 0.1mCi/ml was added to the each wells. After two hours,radioactivity of ³ H-thymidine incorporated into the cells was measuredby a Matrix A counter (manufactured by Packard Co.).

(3) Purification of bOGF-II

i) purification on heparin Sepharose CL-62

The IMR-90 conditioned medium (ca. 90L) sample 1) was filtrated with0.22 μm membrane filter (hydrophilic Milidisk, 2000 cm², manufactured byMilipore Co.), and was divided into three fractions. Each fraction wasapplied to haparin Sepharose CL-6B (5×4.1 cm) (80 ml) equilibrated with10 mM Trls-HCI 0.3 M NaCl pH 7.5. After washing with tomM Triso-HCl, pH7.5 at a flow rate of 500 ml/hr., a heparin Sepharose CL-6B adsorbentprotein fraction was eluted with l-mM Tris-HCu, 2M NaCl, pH 7.5. Thisfraction was designated as sample 2.

ii) purificatin on HiLoad-Q/FF

The heparin Sepharose adsorbent fraction (sample 2) was dialyzed against10 mM Tris-HCl, pH 7.5, supplemented with CHAPS to the finalconcentration of 0.1%, incubated at 4° C. overnight, and divided intotwo fractions. Each fraction was then applied to an anion-exchangecolumn (HiLoad-Q/FF, 2.6×10 cm, manufactured by Pharmacia Co.)equilibrated with 50mM Tris-HCl, 0.1% CHAPS, pH 7.5 to obtain anon-adsorbent fraction (1000 ml). This fraction was designated as sample3.

iii) purification on HiLoad-S/HP

The HiLoad-Q non-adsorbent fraction (sample 3) was applied to acation-exchange column (HiLoad-S/HP, 2.6×10 cm, manufactured byPharmacia Co.) equilibrated with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5.After washing with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5, the adsorbedprotein was eluted with 0-1M NaCl on a linear gradient over 100 minutesat a flow rate of 8 ml/min. The eluate was fractionated at 12ml/fraction. Each fraction (10 μl) was evaluated for OGF activityaccording to the method in (2). OGF adtivity was found in three peaks(peak 1: bOGF-I, peak 2: bOGF-II, peak 3: bOGF-III). The result wasshown in FIG. 1.

The peak 3 was supposed to be bFGF from the fact that OGF activity inpeak 3 was inactivated by heating at 70° C. for 10 minutes, was elutedwith ca. 1.8 M NaCl from heparin column, and was neutralized byanti-human bFGF antibody.

iv) purification on affinity column (heparin-5PW)

The peak 2 (bOGF-II) fraction (120 ml) was diluted with 240 ml of 50 mMTris-HCl, 0.14 CHAPS, pH 7.5, and applied to an affinity column(heparin-5PW, 0.8×7.5 cm, manufactured by Toso Co.) equilibrated with 50mM Tris-HCI, 0.1% CHAPS, pH 7.5. After washing with 50 mM Tris-HCl, 0.1%CHAPS, pH 7.5, the adsorbed protein was eluted with 0-2 M NaCl on alinear gradient over 60 minutes at a flow rate of 0.5 ml/min. The eluatewas fractionated at 0.5 ml/fraction. Each fraction (2 μl) was evalutedfor OGF activity. A fraction (5 ml) eluted with ca. 1.0-1.2 M NaCl wasfound to have OGF activity and designated as sample 4.

The sample 4 (5 ml) was diluted with 10 ml of 50 mM Tris-HCl, 0.1 %CHAPS, pH7.5, and applied to an affinity column (heparin-5PW, 0.8×7.5cm, manufactured by Toso Co.) equilibrated with 50 mM Tris-HCl₁, 0.1%CHAPS, pH 7.5. After washing with 50 mM Tris-HCl, 0.1% CHAPS, pH 7.5,the adsorbed protein was eluted with 0-2 M NaCl on a linear gradient ata flow rate of 0.5 ml/min. The eluate was fractionated at 0.5ml/fraction. Each fraction (2 μl) was evaluated for OGF activity. Afraction (5 ml) eluted with ca. 1.0-1.2M NaCl was found to have OGFactivity and was designated as sample 5.

The sample 5 (5 ml) was diluted with 10 ml of 50 mM Tris-HCl, 0.1%CHAPS, pH 7.5, and applied to an affinity column (heparin-5PW, 0.5×7.5cm, manufactured by Toso Co.) equilibrated with 50 mM Tris-HCl, 0.1%CHAPS, pH 7.5. After washing with 50 mM Tris-HCl, 0.2 M NaCl, 0.1%CHAPS, pH 7.5, the adsorbed protein was eluted with 0.2-0.8 M NaCl on alinear gradient over 60 minutes at a flow rate of 0.5 ml/min. The e.uatewas fractionated at 0.5 ml/fraction. Each fraction (4 μl) was evaluatedfor OGF activity. The result was shown in FIG. 2.

(4) Molecular weight of bOGF-II

The obtained OGF fraction was divided into five fractions at 2ml/fraction (FIG. 2, fractions A-E). Each fraction (100 μl) wassubjected to SDS-polyacrylamide gel electrophoresis under non-reducingcondition. Precisely, each of the fractions A-E (100 μl) was dialyzedagainst water, lyophilized, dissolved in 1.5 μl of a mixture of 10 mMTris-HCl, pH8, 1 mM EDTA, 2.5% SDS, 0.01% bromophenol blue, andincubated at 37° C. overnight. The 1 μl of sample was then analyzed bySDS-polyacrylamide gel electrophoresis with a gradient gel of 8-25%acrylamide (manufactured by Pharmacia Co.) and an electrophoresis device(Fast System, manufactured by Pharmacia Co.). The following molecularweight markers were utilized: phospholipase b (94kD), serum albumin(67kD), ovalbumin (43kD), carbonic anhydrase (30kD), trypsin inhibitor(20.1kD), and a-lactoalbumin (14.4kD). After electrophoresis, proteinbands were visualized by silver stain according to a protocol ofPharmacia Co.. The result was shown in FIG. 3.

Protein band detected at molecular mass of 15kD, was proportional to OGFactivity. The fractions D and E contained only the protein band at 15kD.When this protein was analyzed by SDS-polyacrylamide gel electrophoresisunder reducing condition, a single protein band was detected at almostthe same molecular weight as that obtained under non-reducing condition.

(5) Determination of N-terminal amino acid sequence

The obtained fraction D (500 μl) was applied to a reversed-phase column(BU-300, C4, 2.1×220 mm, manufactured by Applied Biosystems Co.)equilibrated with a mixture of 0.1% luoroacetic acid (TFA), 10%acetonitrile, and eluted on a linear gradient of 10-60% acetonitrileover 50 minutes at a flow rate of 0.2 ml/min., to obtain the desaltedand concentrated sample. This sample was analyzed by protein sequencer(477A-120A, manufactured by Applied Biosystems Co.) for N-terminal aminoacid sequence. The amino acid sequence of the obtained peptide is shownin SEQ ID NO:1. In the sequence, --Xaa-- indicates an amino acid not yetidentified.

(6) Determination of protein amino acid sequence

The fraction C (2000 μl) was concentrated, dissolved with 300 μl of 0.5M Tris-HCl, pH8.5 containing 10 mM EDTA, 7 M guanidine hydrochloride,and dithiothreitol (1 mg), incubated at room temperature for four hours,further supplemented with 2 μl of 4-vinylpyridine, and left in thedarkness at room temperature overnight for pyridylethylation (PE). Tothe sample, 3 μl of 25% TFA was added. The mixture was then applied to areversed-phase column (BU-300, C4, 4.6×30 mm, manufactured by AppliedBiosystems Co.) equilibrated with 10% acetonitrile containing 0.1% TFA,and eluted with 10-50% acetonitrile on a linear gradient over 50 minutesat a flow rate of 1 ml/mln. to obtain reduced PE bOGF-II. A quarter ofthe obtained reduced PE bOGF-II was an alyzed by protein sequencer(477A-120A, manufactured by Applied Biosystems Co.) for N-terminal aminoacid sequence. The obtained amino acid sequence of the peptide is shownin SEQ ID NO. 2.

The residual three quarters of the reduced PE bOGF-II were digested with0.5 μl of endoproteinase Asp-N (manufactured by Boehringer Mannheim Co.)in a mixture (50 μl) of 50 mM phosphate buffer, pH 8.5 containing 1Murea at 37° C. for 15 hours, applied to a reversed-phase column (OD-300,C18, 2.1×220 mm, manufactured by Applied Biosystems Co.) equilibratedwith 0.1% TFA, and eluted on a linear gradient of 0-40% acetonitrileover 80 minutes at a flow rate of 0.2 ml/min.. Elution pattern is shownin FIG. 4. Detected five peaks were analyzed by protein sequencer(477A-120A, manufactured by Applied Biosystems Co.) for N-terminal aminoacid sequence. Each amino acid sequence of the obtained five peptides isshown in SEQ ID NOS:3-7.

(7) Efficacy of byGF-II on the proliferation of MC3T3-El cells.

The fraction D was evaluated for protein concentration according toLowry method by utilizing BSA (bovine serum albumin) as a standard. Thissample was diluted to 100 ng/ml and evaluated for OGF activity, withtwo-fold intervals between contiguous doses, according to the methoddescribed in (2). The result is shown in FIG. 5.

Example 2. Preparation of bOGF-II by application of genetic engineering

(1) Cloning bOGF-II gene

(1) Cloning bOGF-II gene cDNA by PCR

A set of primers for amplifying the tDNA were prepared on the basis ofdetermined amino acid sequence of OGF-II. That is, fromGlu-Thr-Glu-Tyr-Gly-Pro-Cys, (SEQ ID NO:15) an N-terminal amino acidsequence, the DNA sequence coding this was deduced, and mixed primershaving the sequence of 51-GA(A/G)ACNGA(A/G)TA(T/C)GGNCCNTG-3' (SEQ IDNO:16) were synthesized. Here, A/G means A or G; T/C means T or C; and Nrepresents A, G. C or T. From Asp-Lys-Lys-Gly-Phe-Tyr-Lys, (SEQ IDNO:11) an internal amino acid sequence, a DNA sequence coding this wasdeduced, and mixed primers having the complementary sequence chain of5'-TT(A/G)TA(A/G)AANCC(T/C)TT(T/C)TT(A/G)TC-3' (SEQ ID NO:17) weresynthesized. For the synthesis of the primers, DNA Synthesizer 394 ofPerkin Elmer Co. was used. The two kinds of primers (200 pmolrespectively) and single-stranded CDNA derived from Human fetal lungfibroblast IMR-90 polya RNA (1 μg) as the template DNA were used for thepolymerase chain reaction (PCR). The enzyme used was EX Taq(manufactured by Takara Shuzo Co., Ltd.) The reaction solution contained5 μl of 10×ExTaq buffer, 4 μl of 2.5 mM DNTP, 1 μl of the CDNA solution,0.25 μl of Ex Taq, 29.75 μl of distilled water, and each 5 μl of theprimer (40 μM) in a final volume of 50 μl. The reaction condition is asfollows. After keeping the reaction mix at 95° C. for 3 minutes, 30cycles of three-step incubation were performed that consisted of 95° C.for 3 minutes, 50° C. for 30 seconds, and 70° C. for 2 minutes. Afterthese reactions, the reaction mix was kept at 70° C. for 5 minutes.After the reaction was completed, 8 μl of the reaction solution wassubjected to 4% agarose gel electrophoresis; several bands includingabout 120 bp fragment were detected. The reaction solution in a volumeof 4.5 μl, PCRII (original TA cloning kit, Manufactured by InvitrogenCo.) cloning vector in a volume of 0.5 μl, and DNA ligation kit (version2) liquid 1 (manufactured by Takara Shuzo Co. Ltd.) in a volume of 5 μlwere mixed and kept at 16° C. overnight. Using 5 μl of the ligationreaction solution, E. coli DH5α (prepared by BRL Co.) was allowed totransformation. The length of fragments inserted in the plasmid harboredinserted in the resultant ampicillin resistance bacteria was measured byPCR, and 4 strains having about 120 bp inserted fragment were isolated.In the reaction, the two kinds of primers mentioned above were used.From the 4 isolated strains, plasmid DNA was purified, and the DNAsequences of the inserted fragments were determined. When the amino acidsequence deduced from the nucleotide sequence was analyzed, all theclones had a reading frame that coincided with the amino acid sequencedetermined from OGF-II protein. One clone (Clone #1) was subjected tothe following experiment.

(2) Cloning OGF-II full length CDNA

The plasmid of Clone #1 was purified, digested by a restriction enzymeEcoRI (Prepared by Takara Shuzo Co. Ltd.), and subjected to agarose gelelectrophoresis, and OGF-II CDNA of about 120 bp was isolated. Thisfragment was labeled with ³² P using Megaprime kit (manufactured byAmersham Co.).and α³² P-dCTP and served as the probe in the followingexperiment. From Human fetal lung fibroblast IMR-90 polyA⁺ RNA (5 μg),double-stranded cDNA was-synthesized according to the manual of GreatLengths cDNA Synthesis kit (manufactured by Clontech Co.). The polyA⁺RNA was prepared by Fast Track (manufactured by Stratagene Co.). Themethod for synthesizing the double-stranded CDNA is briefly describedbelow. The polyA⁺ RNA (5 μg) and Oligo (dT)₂₅ (dN) primer were mixed andmade into a final volume of 12.5 μl with addition of distilled water,and the solution was kept at 70° C. for 3 minutes and allowed to cool inice for 2 minutes. Into this solution, 3.2 μl of distilled water, 5 μlof 5×First-strand buffer, 0.5 μl of DTT (dithiothreitol), 1.3 μl of dNTP(20 mM each) and 2.5 μl (500 units) of MMLV (RNaseH⁻) were added, andthe mixture was kept at 42° C. Further, 123.5 μl of distilled water, 40μl of 5×second-strand buffer, 1.5 μl of dNTP (20 mM each) and 10 μl ofSecond-strand enzyme cocktail were added therein, and the mixture waskept at 16° C. for 2 hours. Into this reaction solution, 15 units of T4polymerase was added; the resultant was kept at 16° C. for further 30minutes and the reaction was stopped with addition of 10 μl of 0.2 MEDTA followed by chloroform and isoamyl alcohol treatments and ethanolprecipitation. To the terminal of this double-stranded cDNA,EcoRI-SalI-NotI linker (prepared by Clontech Co.) was attached. Then,the resultant was inserted into ZAP Express phage (prepared byStratagene Co.) DNA that was preliminarily cut with a restriction enzymeEcoRI and treated with CIAP (bovine tetis alkaline phosphatase). Therecombinant DNA obtained was subjected to packaging, and infected to E.coli XL1-Blue MRF' (prepared by Stratagene Co.); plaques were formed onNZY agar medium (0.5% NaCl, 0.2% MgSO₄ ·7H₂ O, 0.5% Yeast Extract, 1% NZamine, pH 7.5, 1.5% agar). For the packaging, Gigapack II Gold packagingextract (prepared by Stratagene Co.) was used. The phage formed on theagar medium was -transferred onto nylon membrane, Hybond-N (manufacturedby Amersham Co.) and the phage DNA was fixed. The resultant membrane wasimmersed into hybridization buffer that contained 100 μg/mL of salmonsperm DNA (manufactured by Amersham Co.) and treated for 4 hours at 65°C., and immersed into the mentioned buffer that contained heat-denatured³² p labeled DNA probe (2.5×10⁵ cpm/ml) mentioned above at 65° C.overnight to allow hybridization. After the membrane was washed, the 3clones that had OGF-II CDNA were able to be selected from about onemillion phages. The 3 clones were purified through two more screenings.After XL1-Blue MRF'cells, an E. coli strain, were infected with thepurified phages, the infected cells were co-infected with helper phageExAssist (prepared by Stratagene Co.). The supernatant cultivate wasused to infect E. coli XLOLR (prepared by Stratagene Co.), and E. colicells that became kanamycin resistant were obtained. The structure ofplasmid DNA in one of these E. coli clones was analyzed and thenucleotide sequence of the inserted fragment was determined. Thesequence is shown as SEQ ID NO:8. The amino-acid sequence deduced fromSEQ ID NO:8 of OGF-II is shown as SEQ ID NO:9. Comparing this amino-acidsequence with wild type OGF-II amino-acid sequence, it is found that theformer has one more Lys amino acid at the C-terminal (85th amino acid).Accordingly, the C-terminal lysine of bOGF-II purified form IMR-90 cellsconditioned medium. Accordingly, the C-terminal lysine of bOGF-IIpurified from IMR-90 cells conditioned medium may have been cleaved offby (a) carboxypeptidase(s). This means that all of the 3 clonescontained the DNA that coded an open reading frame consisting of 85amino acids. from OGF-II N-terminal Glu-Thr-Glu-Tyr (SEQ ID NO:18). Theopen reading frame was determined from the position of the firsttermination codon and the amino acid sequence from the OGF-II protein.One of the obtained plasmid is named PBK-CMV OGF-II (3).

(2) Expression of OGF-II CDNA in E. Coli

The full length of OGF-II CDNA was amplified by PCR and isolated. Theprimers used were Q30F 5'-GGGGATCCGAGACAGAATATGGTC-3' (SEQ ID NO:19) andQ30R 5'-CCAAGCTTCTACTTGCTCTGCATACT-3'(SEQ ID NO:20). These primers weredesigned so that the amplified products can be digested with restrictionenzymes BamHI and HindIII. Using the template of 20 ng of pBK-CMV OGF-II(3) and the primers (Q30F and Q30R), PCR was performed. The reactionsolution contained 10 μl of 10×ExTaq buffer, 8 μl of 2.5 mM DNTP, 0.5 μlof Ex Taq, 9.5 μl of the DNA solution, 70 μl of distilled water, and 1μl of the primer (100 μM each) in a final volume of 100 μl. Afterkeeping the reaction mix at 95° C. for 3 minutes, 25 cycles ofthree-step incubation were performd that consisted of 95° C. for 3minutes, 50° C. for 30 seconds, and 70° C. for 2 minutes. After thesereactions the reaction mix was kept at 70° C. for 5 minutes. After theamplification, the primers were removed with Microcon 100 (Amicon Co.)and the PCR product was digested with restriction enzymes BamHI andHindIII; thereafter, the product was mixed with 20 ng of pOE30 (preparedby QIAGEN Co.) that was preliminarily cut with BamHI and HindIII andused for the ligation. Using the ligation solution, E. coli XL2-Blue(prepared by Stratagene Co.) was transformed. From the obtainedampicillin resistant colonies, a clone having the target insert fragmentwas separated by analyzing the DNA fragments digested with restrictionenzymes and sequencing. The plasmid in this clone is named pQE30-OGF-II.The E. coli XL2-Blue (pQE30-OGF-II) having this plasmid has beendeposited to NIBH, Agency of Industrial Science and Technology as "FERMBP-5139". The structure of plasmid contained in the deposited E. coli isshown FIG. 6. This strain was cultivated with shaking in super medium(2.5% bacto-tryptone, 1.5% bacto-yeast extract, 0.5% NaCl, 50 μg/mlampicillin); when OD600 nm became 0.8, isopropylβ-D-thio-galatopylanoside (IPTG) was added at a final concentration of0.5 mM, and the shaking cultivation was continued for another 20 hoursto produce OGF-II with the E. Coli. After the cultivation finished, thestrain was collected; the main band of molecular weight of about 15 KDwas confirmed with SDS polyacrylamid gel electrophoresis. This molecularweight well coincided with that of the OGF-II that was obtained fromcultured supernant of IMR90.

(3) Purification of His6-bOGF-II

To test the osteoblast growth activity of the protein obtained bytranslating bOGF-II gene, expression of the gene was performed usingOIAexpress Kits (manufactured by QIAGEN Co.). In this system, proteincontaining histidine hexamer tag is produced, and purification is doneusing nickel-chelating nitrilotriacetic acid resin column that has highaffinity to hlstidine hexamer tag. This system is generally used forefficiently purifying expressed protein. By using this system, bOGF-IIis produced in the form having cluster of six histidines atthe,N-terminal (His6-bOGF-II). To 0.35 g of E. coli treated with IPTG,1.75 ml of 10 mM Tris-HC1, pH 8.0 that contains 8 M urea and 0.1 Msodium phosphate was added; agitated for one hour at room temperature.After the agitation, the sample was conttifuged at 12,000 rpm, for 15minutes at room temperature and the supernant was collected. Thesupernant was added to 8 ml of 50% suspension of nickel-chelatingnitrilotriacetic acid resin (manufactured by QIAGEN Co.) that wasequilibrated with 10 mM Tris-HC1, 8 M urea, 0.1 M sodium phosphate, pH8.0, the mixture was agitated for 45 minutes at room temperature. Thisresin was transferred into a column of 1.6 cm inside diameter and washedwith 20 ml of 10 mM TrisHCl, 8 M urea, 0.1 M sodium phosphate, pH 8.0,at a flow rate of about 0.5 ml/min. Then, this column was further washedwith 10 mM Tris-HCl, 8 M urea and 0.1 M sodium phosphate, pH 6.3, at aflow rate of about 0.5 ml/min. Finally, the target His6-bOGF-II waseluted with 10 mM Tris-HCl, 250 mM ^(i) midazole, 8 M urea, 0.1 M sodiumphosphate, pH 6.3 at a flow rate of about 0.5 ml/min. The purifiedHis6-bOGF-Il fraction was dialyzed for phosphate buffered saline.

(4) Osteoblast growth activity of His6-bOGF-II

The osteoblast growth activity of His6-bOGF-II was tested as describedin Example 1 (2), method for testing ostsoblast growth activity. Thatis, the purified His6-bOGF-Il fraction described above was added to theassay medium, and incorporation of ³ H-thymldine to mouse osteoblastMC3T3-El cells was tested.. The results are shown in FIG. 7. From theseresults, it has been confirmed that the protein produced by translatingbOGF-II gene has the osteoblast growth activity as the wild type bOGF-IIhas.

Possibility for Use in Industry

According to the invention, a novel.protein with osteoblast growthactivity and methods for producing the protein are provided. Since theprotein of the instant invention has osteoblast growth activity, it isuseful as a drug for the treatment of bone mass reduction such asosteoporosis and as antigen for immunological diagnosis of the diseases.

Reference to Microoraanisms deposited

Deposited Organization and Address that the Organisms were deposited:

National Institute of Bioscience and Human-Technology

Agency of Industrial Science and Technology

Ministry of International Trade and Industry

1-3, Higashi 1-chome, Tsukuba City, Ibaragi Prefecture

Date of the Deposition to the Deposited Or ganimation:

Jun. 19, 1995

Deposit Number that the Deposited Organization provided:

FERM BP-5139

This deposit was transf erred from the national deposit (AcessionNumber: FERM P-14942 of May 26, 1995) on Jun. 19, 1995.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:20                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Glu Thr Glu Tyr Gly Pro Xaa Arg Arg Glu Me - #t Glu Asp Thr Leu Asn       1               5  - #                 10 - #                 15              - - His Leu Lys Phe Leu Asn Val Leu Ser                                                   20     - #             25                                         - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Me - #t Glu Asp Thr Leu Asn       1               5  - #                 10 - #                 15              - - His Leu Lys Phe Leu Asn Val Leu Ser Pro Ar - #g Gly Val Xaa Ile Pro                   20     - #             25     - #             30                  - - Asn Cys Xaa Lys Lys Gly Phe Tyr                                                   35         - #         40                                             - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Asp Val His Cys Tyr Ser Met Gln Ser                                       1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Me - #t Glu                       1               5  - #                 10                                     - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Asp Lys Tyr Gly Gln Pro Leu Pro Gly Tyr Th - #r Thr Lys Gly Lys Glu       1               5  - #                 10 - #                 15              - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:6:                                - - Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cy - #s Arg Pro Ser Lys Gly      1               5   - #                10  - #                15              Arg Lys Arg Gly Phe Cys Trp Cys Val                                                       20      - #            25                                          - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Asp Thr Leu Asn His Leu Lys Phe Leu Asn Va - #l Leu Ser Pro Arg Gly       1               5  - #                 10 - #                 15              - - Val His Ile Pro Asn Cys                                                               20                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 258                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA to mRNA                                      - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: human                                                  - -     (ix) FEATURE:                                                                  (B) LOCATION: 1 to 2 - #58                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GAGACAGAAT ATGGTCCCTG CCGTAGAGAA ATGGAAGACA CACTGAATCA CC -             #TGAAGTTC   60                                                                   - - CTCAATGTGC TGAGTCCCAG GGGTGTACAC ATTCCCAACT GTGACAAGAA GG -            #GATTTTAT  120                                                                   - - AAGAAAAAGC AGTGTCGCCC TTCCAAAGGC AGGAAGCGGG GCTTCTGCTG GT -            #GTGTGGAT  180                                                                   - - AAGTATGGGC AGCCTCTCCC AGGCTACACC ACCAAGGGGA AGGAGGACGT GC -            #ACTGCTAC  240                                                                   - - AGCATGCAGA GCAAGTAG             - #                  - #                     258                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 85                                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Me - #t Glu Asp Thr Leu Asn       1               5  - #                 10 - #                 15              - - His Leu Lys Phe Leu Asn Val Leu Ser Pro Ar - #g Gly Val His Ile Pro                   20     - #             25     - #             30                  - - Asn Cys Asp Lys Lys Gly Phe Tyr Lys Lys Ly - #s Gln Cys Arg Pro Ser               35         - #         40         - #         45                      - - Lys Gly Arg Lys Arg Gly Phe Cys Trp Cys Va - #l Asp Lys Tyr Gly Gln           50             - #     55             - #     60                          - - Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Gl - #u Asp Val His Cys Tyr       65                 - # 70                 - # 75                 - # 80       - - Ser Met Gln Ser Lys                                                                       85                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Glu Thr Glu Tyr Gly Pro Cys                                               1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Asp Lys Lys Gly Phe Tyr Lys                                               1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:12:                               - - Glu Thr Glu Tyr                                                           - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pair                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: genomic DNA                                       - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:13:                               - - GGGGATCCGA GACAGAATAT GGTC          - #                  - #                  24                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pair                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: genomic DNA                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - CCAAGCTTCT ACTTGCTCTG CATACT          - #                  - #                26                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Glu Thr Glu Tyr Gly Pro Cys                                               1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pair                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: genomic DNA                                       - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:16:                               - - AGRACNGART AYGGNCCNTG            - #                  - #                     20                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7                                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Asp Lys Lys Gly Phe Tyr Lys                                               1               5                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pair                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: genomic DNA                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - TTRTARAANC CYTTYTTRTC            - #                  - #                     20                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pair                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: genomic DNA                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - GGGGATCCGA GACAGAATAT GGTC          - #                  - #                  24                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pair                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: genomic DNA                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - CCAAGCTTCT ACTTGCTCTG CATACT          - #                  - #                26                                                                      __________________________________________________________________________

We claim:
 1. A purified protein with an activity to stimulate osteoblastcell growth, having the amino acid sequence as set forth in SEO ID NO:9.
 2. A purified protein with an activity to stimulate osteoblast cellgrowth, wherein the protein comprises the amino acid sequence as setforth in SEQ ID NO:9.
 3. A method for producing a protein comprising:cultivating human fibroblasts; passing a conditioned medium thereof overa heparin column; eluting an adsorbed fraction; passing the eluant overan anion exchange column to obtain the non-adsorbed fraction; passingthe non-adsorbed fraction over a cation exchange column; and furtherpurifying with a heparin column to produce a protein that has thefollowing characteristics:(a) a molecular weight of about 15 kD underreducing and non-reducing conditions by sodium dodecylsulfate--polyacrylamide gel electrophoresis (SDS-PAGE), (b) a highaffinity to a cation exchanger and heparin, and (c) an activity tostimulate osteoblast cell growth that is decreased by heating at 70° C.for 10 minutes and inactivated by heating at 90° C for 10 minutes. 4.The method for producing a protein according to the claim 3, wherein theobtained protein comprises the amino acid sequence as set forth in SEQID NO:9, and has an activity to stimulate osteoblast cell growth.
 5. Ahost cell which is transformed by an expression vector containing cDNAwhich encodes the amino acid sequence set forth in SEQ ID NO: 9, andwhich is Escherichia coli pQE30-OGF11 (Ferm BP-5139).